Abstract

A series of N-substituted and N′-substituted aminothiazole-derived morphinans (5) were synthesized for expanding the structure-activity relationships of aminothiazolo-morphinans. Although their affinities were somewhat lower than their prototype aminothiazolo-N-cyclopropylmorphinan (3), 3-aminothiazole derivatives of cyclorphan (1) containing a primary amino group displayed high affinity and selectivity at the κ and μ opioid receptors. [35S]GTPγS binding assays showed that the aminothiazolomorphinans were κ agonists with mixed agonist and antagonist activity at the μ opioid receptor. These novel N′-monosubstituted aminothiazole-derived morphinans may be valuable for the development of drug abuse medications.

Introduction

The opioid system modulates several key physiological and behavioral processes, such as: pain perception, the stress response, the immune response, and neuroendocrine function.1 With the discovery of the three different opioid receptors (κOR, μOR, and δOR), different functions and effects of the three receptor subtypes have been elucidated. Notably, it was found that the κ opioid receptor plays a role in the development of drug addiction, specifically by altering the dopamine reward pathway. Thus, the κ receptor has been implicated as a primary target for the development of pharmacotherapies for the treatment of cocaine dependence.2,3 Recent behavioral studies suggested that κ/μ opioids may be useful for the treatment of cocaine abuse and dependence.4 We reported that both acute and chronic treatment with mixed κ/μ opioids cyclorphan (1)5 and butorphan,5, 6 reduced cocaine self-administration dose-dependently and produced fewer side-effects than κ-selective agonists.7 However, the opioid derivatives are not metabolically stable: the free phenolic hydroxyl group in cyclorphan (1) and butorphan is also a potential site for metabolism, conjugation, and excretion, resulting in low oral bioavailability and short duration of action.1,8, 9 In an attempt to further extend the duration of action and to manipulate relative affinity and efficacy at κOR, modification of the phenolic hydroxyl group of cyclorphan has been performed, by incorporating 3-amino (2) 10, 3-aminothiazole (3, ATPM) 10, 2-aminooxazole (4) 11 isosteres (Figure 1).

Among this series, one compound, 3 (Figure 1), has been identified to possess high affinity at κOR (Ki = 0.049 nM), and mixed κ agonist and μ-agonist/antagonist.10c (Table 1). Previous studies have shown that 3 inhibited morphine-induced antinociceptive tolerance, with less potential to develop tolerance and reduce heroin self-administration with lower sedative effect.12 However, recent in vivo studies of 3 in mice in the 55°C tail-flick test showed that this compound does not appear to have a longer duration of action than the phenolic compound 1.13 Aiming to extend duration of action and to improve oral bioavailability, a structure-activity relationship (SAR) study has been conducted to investigate the effect of modifications of N-substituent (R3) and N′-3-amino-substituted (R1, R2) of the morphinan 5 (Figure 1).

Herein we report the synthesis and pharmacological evaluation of a series of N-substituted (R3) and N′-3-amino-substituted (R1, R2) analogs of morphinan. The highly potent (−)-3-hydroxy-N-(E)-iodoallylmorphinan14 suggested the replacement of the N-cyclopropylmethyl group in cyclorphan with a fluoropropyl group to make compounds 7c and its analogs 9c, 11, 13c, and 19c, and introduced a trifluoroethyl substituent to the amino group of the aminothiazole component in 3 to make compound 15 (Scheme 1 and Scheme 2).

Chemistry

The synthesis of all target compounds was initiated from commercially available levorphanol tartrate, which, after conversion to its free base, could be demethylated to norlevorphanol (6). Next, 6 was alkylated with either cyclopropylmethyl bromide, cyclobutylmethyl bromide, fluoropropyl bromide, or (−)-(s) tetrahydrofurfuryl (R)-camphor-10-sulfonate to yield 7a–d, respectively. Subsequent triflation of morphinans 7a–d afforded triflates 8a–d, which were subjected to palladium-catalyzed amination to afford amines 9a–d in moderate yields. The aminothiazoles 3, 10–12 were then synthesized in 55–61% yield according to literature procedure (Scheme 1).6, 10

For the synthesis of N′-methyl substituted aminothiazolomorphinans 13a–c, aminothiazolomorphinans 3, 10 and 11 were formylated with freshly prepared formyl acetate (prepared by heating a mixture of HCOOH and Ac2O), followed by reduction, yielding the novel N′-methyl-3-aminothiazolomorphinans 13a–c in 34–45% yields (Scheme 2).15 Treatment of 13a and 13c with paraformaldehyde and NaBH4 yielded dimethyl substituted aminothiazolomorphinans 17a and 17c in 83–89% yields.16 N′-Trifluoroethyl derivative 15 was prepared in 45% yield by treating 3 with trifluoroacetic anhydride in the presence of Et3N, followed by reduction.17 N′-ethyl substituted aminothiazolomorphinans 19a–c were prepared analogously 17 in which compound 3, 10, and 11 were first acylated and then reduced. Treatment of 13a and 19a with acetic anhydride produced N′-disubstituted derivatives 14 and 20. Compounds 3 and 11 were also condensed with propionaldehyde, followed by reduction of the resulting imines to N′-propyl substituted aminothiazolomorphinans 16a and 16c in 47–55% yields18 (Scheme 2).

Using literature procedures,193 was reductively aminated to afford 21 and 22 in 65–68% yields. Methoxybenzylated derivative 21 was demethylated with BBr3 to give 23 in 74% yield.20 Furthermore, 3 was treated with EtSCN to yield thiourea 24 in 45% yield (Scheme 3).21

For preparation of aryl substituted derivatives 26 and 27, 3 was converted to 3-bromothiazolo-N-cyclopropylmethylmorphinan 25 through the Sandmeyer reaction.22 Compound 25 was treated with aniline and 2-aminopyridine, respectively, to yield 26 and 27 in 70–72% yields. Treatment of 25 with piperazine produced 28 in 63% yield (Scheme 4). 23

Previous reports from our laboratories indicated that changing N-substituted group (R3) in the aminothiazolomorphinan drastically altered potency and efficacy. Compared to the N-methyl derivative 29, the N-cyclopropyl compound 3 displayed a much higher (130-fold) affinity at κ the receptor. From the data shown in Table 1, the N-fluoropropyl derivative 11 had high affinity at κ (0.30 nM) and good selectivity for κ over μ (9-fold) and δ (180-fold) receptor. N-Tetrahydrofurylmethylmorphinan 12 also showed high affinity at κ (0.83 nM) and moderate affinity at μ (2.4 nM). Introducing a small alkyl group at N′, 13a had similar affinity with 3 at the κ receptor, with Ki value of 0.066 nM. Compound 13a also displayed high selectivity for κ over μ (45-fold) and δ (380-fold) receptors. When the size of alkyl group at N′ increased, we observed a smooth decrease in affinity at κ and μ receptors in 19a, 15, 16a. However, they still displayed high affinity at κ (Ki = 0.15–1.6 nM). N′-Acetyl aminothiazolomorphinan 18a showed low affinity at κ (13 nM) and μ (57 nM), perhaps due to the lowered basicity of nitrogen in this analogue. When the N′-substituent on amine was either benzyl (22), 3-OH-benzyl (23), or 3-MeO-benzyl (21), binding affinities were low [Ki = 2.1–4.8 nM (κ) and Ki = 9.1–10 nM (μ)]. Analogues 26 and 27, which contained N′-aryl and (hetero)aryl groups, were prepared. Compared to the alkyl substituted aminothiazole analogues (13a, 15, 19a), an unexpected decrease of affinity at κ and μ receptors was observed in 26 and 27. The N′-piperazine substituted aminothiazolomorphinan displayed very low affinity at κ (110 nM) and at μ (2700 nM). To explore the possibility that incorporation of an additional polar group into the N′-substitution would further enhance affinity, we prepared a thiourea analogue as a probe. However, the N′-ethylthiourea analogue displayed low affinity at κ (18 nM) and at μ (130 nM).

It was found that N′-disubstituted aminothiazolo-N-cyclopropylmorphinans generally had lower affinity when compared to N′-monosubstituted- aminothiazolo-N-cyclopropylmorphinans. N′-Dimethyl substituted derivative (17a) was the most potent compound in the series of N′-disubstituted compounds synthesized, with an affinity of 0.45 nM at the κ receptor and 10 nM at the μ receptor. Ki values for N′-methyl derivative (13b) and N′-ethyl derivative (19b) were 2.4 and 3.7 nM for κ, respectively. N-fluoropropyl N′-methyl (13c) and N′-ethyl (19c) morphinan analogues were very potent, with Ki values being <1 nM for binding to the κOR. N-fluoropropyl N′-propyl (16c) and N′-dimethyl (17c) morphinan analogues showed low affinity at the κ and μ receptors. From a SAR perspective, the binding affinities of substituted aminothiazolomorphinan analogues at all three receptors were generally lower than the binding affinities of the aminothiazole precursors (3, 10, 11). However, most of the N′-monosubstituted analogues showed high affinities at κ (Ki = 0.06–0.94 nM).

To characterize the relative efficacy of these ligands, 1, 3, and 10, were selected for the [35S]GTPγS assay. The stimulation and inhibition of [35S]GTPγS binding mediated by κ and μ opioid receptors are shown in Table 2 and Table 3, respectively.

Agonist and antagonist properties of compounds in stimulating [35S]GTPγS binding mediated by the μ opioid receptora

These ligands produced maximal stimulation of [35S]GTPγS binding (Emax) at κ comparable to that of ligand 3. Ligands 13c, 15, 19a, and 19c produced a higher Emax than that of selective agonist U50,488. None of these compounds inhibited U50,488-stimulated [35S]GTPγS at κ, demonstrating that all of these ligands were full κ agonists.

From the data shown in Table 3, ligands 11, 13a, and 17a displayed partial agonist activity at μ receptor. Ligands 12, 13c, 19a and 19c showed full agonist activity at the μ receptor; they did not inhibit DAMGO-stimulated [35S]GTPγS binding.

Conclusion

We have extended the structure-activity relationships of aminothiazolomorphinans by introducing different groups to N- and N′-positions. A series of aminothiazolomorphinans and their N′-mono- and di-substituted derivatives were synthesized, and their pharmacological properties at opioid receptors were evaluated. It was found that substituents at the aminothiazole nitrogen tended to reduce the affinity of the compounds, with the exception of the methyl group (13a), which retained high affinity at the κ receptor (0.066 nM) as well as good selectivity for κ over μ (45-fold) and δ (380-fold) receptor. N′-disubstituted aminothiazolo-N-cyclopropylmorphinan analogues 17a, 14, 20, and 17c had lower affinity at all three opioid receptors. However, N′-dimethyl aminothiazolo-N-cyclopropylmorphinan 17a was also a potent and selective compound, with an affinity of 0.45 nM at the κ receptor and 10 nM at the μ receptor. The same pattern was observed with the replacement of the cyclopropylmethyl group in 1 with the fluoropropyl group. 13a, 19a, and 17a may prove to be useful for the potential development as medications for cocaine or opioid abuse. The [35S]GTPγS binding assay revealed that all new compounds were full agonists at the κ receptors, ligands 11, 13a, and 17a were partial agonists at the μ receptors, and ligands 12, 13c, 19a, and 19c were full agonists at the μ receptors. Preliminary evaluation of 3 in non-human primates reduced self-administration and attenuated food intake, probably due to its kappa agonist properties.24

Experimental Section

General Synthetic Methods

1H (and 13C NMR) spectra were recorded at 300 MHz (75 MHz) on a Varian Mercury 300 spectrometer. Chemical shifts are given as δ value (ppm) downfield from tetramethylsilane as an internal reference. Melting points were determined on a Thomas-Hoover capillary tube apparatus and are reported uncorrected. Elemental analyses, performed by Atlantic Microlabs, Atlanta, GA, were within (0.4% of theoretical values. Analytical thin-layer chromatography (TLC) was carried out on 0.2 micrometer Kieselgel 60F-254 silica gel plastic sheets (EM Science, Newark, NJ). Flash chromatography was used for the routine purification of reaction products. Eluent systems are described for the individual compounds.

General Procedure6 for the Preparation of 3-Hydroxy-N-alkyl-morphinans 7a–d

The mixture of norlevorphanol (5 mmol), K2CO3 or NaHCO3 (7.5 mmol), and either bromomethyl cyclopropane, bromomethyl cyclobutane, 1-bromo-3-fluoropropane, or (S)-tetrahydrofurfuryl (1R)-camphor-10-sulfonate (7.5 mmol) in 20 mL anhydrous DMF were stirred at 90–95 °C for overnight. After the reaction was judged complete by TLC, the reaction mixture was cooled, poured into water, extracted with CHCl3. The organic phase washed by brine, dried over anhydrous Na2SO4, and concentrated in vacuo to give crude product, purified by flash silica gel column (DCM: MeOH = 20:1 – 5:1) to give the corresponding morphinans 7a–d. The analytical data for 7a–b, 7d was in agreement with literature values. 6

3-Hydroxy-N-alkylmorphinan 7a–d (3.5 mmol), was dissolved in anhydrous DCM (20 mL) and Et3N (3.5 mL). The mixture was cooled to 0 °C, and then PhNTf2 (1.94 g, 5.4 mmol) was added. The mixture was allowed to warm to rt overnight. The solution was diluted with DCM (40 mL), washed with 1N HCl followed by brine, and then dried with anhydrous Na2SO4. The solvent was removed in vacuo to afford the crude product, which was purified by flash silica gel column to give corresponding triflates. The analytical data for 8a–b was in agreement with literature values. 6

General Procedure6,10 for the Preparation of Aminothiazolomorphinans 3, 10-12

The amine (1.1 mmol) and KSCN (426 mg, 4.4 mmol) were dissolved in 10 mL glacial acetic acid. A solution of Br2 (180 mg, 1.1 mmol) in 2 mL of glacial acetic acid was added dropwise. The mixture was stirred for 48h, then basified with 10% NaOH and extracted with CHCl3. The organic layer was washed with brine, dried over anhydrous Na2SO4 and concentrated in vacuo. The crude product was purified by flash silica gel column to yield corresponding aminothiazole. The analytical data for 3, 10 was in agreement with literature values. 10c

General Procedure for Synthesis of N′-Methyl-aminothiazolomorphinans 13a–c

At room temperature and under nitrogen atmosphere, freshly made HCOOAc (0.7 ml, 5.0 mmol, this reagent was prepared by heating a mixture of 1.8 mL HCOOH and 3.8 mL HOAc at 50 °C for two hours) was slowly added to a solution of 3-aminothiazolomorphinan (0.88 mmol). The mixture was stirred at room temperature for 24h. The resulting mixture was then concentrated to dryness and directly separated by flash silica gel column to give the intermediate formate. The intermediate (0.65 mmol) was dissolved in 5 mL anhydrous THF followed by addition of LiAlH4 (50 mg, 1.3 mmol, added in one portion at 0 °C). Then resulting suspension was stirred at room temperature for 16h. After reaction was judged to be complete by TLC, 1 mL of water was added slowly to quench the reaction and followed by addition of 1 mL aqueous 2 N NaOH. The resulting olution was diluted with 50 mL of CH2Cl2 and washed with water and brine. The organic layer was dried over anhydrous Na2SO4 and concentrated in vacuo. The crude product was purified by flash silica gel column to give corresponding morphinans.

General Procedure for Synthesis of N′-Propyl-aminothiazolomorphinans 16a and 16c

The mixture of aminothiazolomorphinan (0.3 mmol), proponialdehyde (43 μL, 0.6 mmol) in 2 mL MeOH was stirred at 60 °C for overnight. The reaction was judged to be complete by TLC. Next, NaBH4 (45.6 mg, 1.2 mmol) was added, and the resulting mixture was stirred at 60 °C for 8h. After this period, the solvents were removed in vacuo. The residue was then directly purified by flash silica gel column to give corresponding morphinans.

General Procedure for Synthesis of N′, N′-Dimethyl-aminothiazolomorphinans 17a and 17c

To a stirred mixture of N′-methyl-aminothiazolomorphinan (0.15 mmol), paraformaldehyde (44 mg, 1.5 mmol), and NaBH4 (28.8 mg, 0.76 mmol) in THF (3 mL) at rt under nitrogen atmosphere was added dropwise trifluoroacetic acid (1.5 mL). The resulting mixture was stirred at rt for 24h, then poured into a mixture of 25% aqueous NaOH (5 mL) and ice to make strongly alkaline solution, which was then diluted with saturated NaCl solution (5 mL), and extracted with CH2Cl2. The combined extracts were dried by anhydrous Na2SO4, filtered, and concentrated in vacuo to afford a yellow solid. The solid was treated with 10% HCl. The aqueous layer was washed with CH2Cl2, and then added 10% NaOH to make the free base. The resulting aqueous layer was extracted with CH2Cl2. The combined extracts were washed with brine, and then dried by anhydrous Na2SO4, filtered, and concentrated in vacuo to give corresponding morphinans.

General Procedure for Synthesis of N′-Acetyl-aminothiazolomorphinans 18a and 18c

The mixture of aminothiazolo-morphinan (0.41 mmol), pyridine (2.1 mL), and acetic anhydride (1.1 mL) was stirred at room temperature for 24h. The volatile components were removed in vacuo. The residue was purified by flash silica gel column to afford the corresponding morphinans.

General Procedure for Synthesis of N′-Ethyl-aminothiazolomorphinans 19a -c

At room temperature, a solution of N′-acetyl-aminothiazolomorphinan (N′-acetyl-2′-aminothiazolo-N-cyclobutylmorphinan 18b was prepared using same procedure with 12a) (0.31 mmol) in 1 mL of dry THF was added to a suspension of LiAlH4 (24 mg, 0.62 mmol) in 2 mL of dry THF. After 24h of stirring, 0.2 mL of water was added to quench the reaction followed by the addition of 0.2 mL of 2 N aqueous NaOH. The resulting mixture was then stirred for 30 min and filtered, and the resulting solid was washed with CH2Cl2. The filtrate was concentrated in vacuo. The resulting residue was then purified by flash silica gel column to afford the corresponding morphinans.

General Procedure for Synthesis of N′-Aryl-aminothiazolomorphinans (26–27) and 3-(piperazin-1-yl)-thiazolo[5,4-b]-N-cyclopropylmethylmorphinan (28)

To a solution of either aniline, 2-aminopyridine or piperazine (1.32 mmol) in dry THF (4 ml), NaH (55 mg; 1.32 mmol, ω = 0.6) was added and stirred for 30 minutes at 50 – 60 °C. Next, 2′-bromo-thiazolo[5,4-b]-N-cyclopropylmethyl-morphinan 25 (137 mg, 0.33 mmol) was added and the reaction mixture was left to stir for 4 hours. The reaction mixture was then concentrated in vacuo. The residue was then dissolved in CH2Cl2 and washed with water and brine. The organic layer was dried over Na2SO4, filtered, concentrated. The crude product was purified by flash silica gel column to give the corresponding morphinans 26–28.

Opioid binding to the human κ, δ, and μ opioid receptors

Chinese hamster ovary (CHO) cells stably transfected with the human κ opioid receptor (hKOR-CHO), δ-opioid receptor (hDOR-CHO) were obtained from Dr. Larry Toll (SRI International, Palo Alto, CA), and the μ-opioid receptor (hMOR-CHO) were obtained from Dr. George Uhl (NIDA Intramural Program, Baltimore, MD). The cells were grown in 100-mm dishes in Dulbecco’s modified Eagle’s media (DMEM) supplemented with 10% fetal bovine serum (FBS) and penicillin–streptomycin (10,000 U/mL) at 37 °C in a 5% CO2 atmosphere. The affinity and selectivity of the compounds for the multiple opioid receptors were determined by incubating the membranes with radiolabeled ligands and 12 different concentrations of the compounds at 25 °C in a final volume of 1 mL of 50 mM Tris–HCl, pH 7.5. Incubation times of 60 min were used for the κ-selective peptide [3H]DAMGO and the j-selective ligand [3H]U69,593. A 3-h incubation was used with the δ-selective antagonist [3H]naltrindole.

[35S]GTPγS binding studies to measure coupling to G proteins

Membranes from CHO cells stably expressing either the human κ or μ opioid receptor were used in the experiments. Cells were scraped from tissue culture plates and then centrifuged at 1000g for 10 min at 4 °C. The cells were resuspended in phosphatebuffered saline, pH 7.4, containing 0.04% EDTA. After centrifugation at 1000g for 10 min at 4 °C, the cell pellet was resuspended in membrane buffer, which consisted of 50 mM Tris–HCl, 3 mM MgCl2, and 1 mM EGTA, pH 7.4. The membranes were homogenized with a Dounce homogenizer, followed by centrifugation at 40,000g for 20 min at 4 °C. The membrane pellet was resuspended in membrane buffer, and those transfected with the centrifugation step was repeated. The membranes were then resuspended in assay buffer, which consisted of 50 mM Tris–HCl, 3 mM MgCl2, 100 mM NaCl, and 0.2 mM EGTA, pH 7.4. The protein concentration was determined by the Bradford assay using bovine serum albumin as the standard. The membranes were frozen at −80 °C until used.

CHO cell membranes expressing either the human κ opioid receptor (15 μg of protein per tube) or μ opioid receptor (7.5 μg of protein per tube) were incubated with 12 different concentrations of the agonist in assay buffer for 60 min at 30 °C in a final volume of 0.5 mL. The reaction mixture contained 3 μM GDP and 80 pmol of [35S]GTPγS. Basal activity was determined in the presence of 3 μM GDP and in the absence of an agonist, and nonspecific binding was determined in the presence of 10 μM unlabeled GTPγS. Then, the membranes were filtered onto glass fiber filters by vacuum filtration, followed by three washes with 3 mL of ice-cold 50 mM Tris–HCl, pH 7.5. Samples were counted in 2 mL of Ecoscint A scintillation fluid. Data represent the percent of agoniststimulation [35S]GTPγS binding over the basal activity, defined as [(specific binding/basal binding) × 100] − 100. All experiments were repeated at least three times and were performed in triplicate. To determine antagonist activity of a compound at the μ opioid receptors, CHO membranes expressing the μ opioid receptor were incubated with the compound in the presence of 200 nM of the agonist DAMGO. To determine antagonist activity of a compound at the κ opioid receptors, CHO membranes expressing the κ opioid receptor were incubated with the compound in the presence of 100 nM of the κ agonist U50,488.